Number Of Viable Bacteria Research Articles

Akkermansia muciniphila Growth Promoted by Lychee Major Flavonoid through Bacteroides uniformis Metabolism.

Akkermansia muciniphila (A. muciniphila) possesses health-promoting properties. Nevertheless, A. muciniphila enrichment remains a challenging endeavor. Quercetin-3-O-rutinose-7-O-α-l-rhamnoside (QRR), a flavonoid found in lychee pulp, has a unique double-substituted glycosylated structure, requiring a specific intestinal microbiota for effective metabolism. Here, QRR was fermented using a coculture of Bacteroides uniformis and A. muciniphila, and the interactions between the two were elucidated in terms of QRR regulation of microbial growth changes and metabolic properties. The results demonstrated that QRR effectively promoted the proliferation of A. muciniphila based on the metabolic action of B. uniformis in vitro, which was evidenced by a notable increase in the number of viable bacteria. Furthermore, the coculture sample exhibited a significant increase in SCFAs. Qualitative analysis of metabolites by UPLC-ESI-Triple-TOF-MS/MS showed that B. uniformis could release sugars on QRR to produce quercetin-3-O-glucoside-7-O-α-rhamnoside and further quercetin. In the coculture and B. uniformis culture, quercetin was converted to taxifolin, which was identified as a crucial intermediate in the metabolism of QRR. Notably, the metabolite kaempferol was only detected in the coculture. The present study reveals the interaction between QRR and the coculture of A. muciniphila and B. uniformis, providing a practical basis for the potential prebiotic value of QRR.

Leukocyte kinetics and bacterial clearance during S. pneumoniae pneumonia and contributions of ICAM-1.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule that is highly expressed on the pulmonary capillary endothelium, alveolar epithelium and other cell types within the lung. ICAM-1 plays important roles in leukocyte adhesion, migration, and motility. To determine the contributions of ICAM-1 to bacterial clearance and leukocyte kinetics during pneumonia, mice were inoculated with S. pneumoniae and evaluated 1, 4 and 7 days later. Our results show that Icam1-/-mice have a greater number of viable bacteria within the lung at each time point. The impaired clearance observed in Icam1-/- mice was not due to an impediment in leukocyte recruitment. In fact, Icam1-/- mice had a greater number of neutrophils and recruited inflammatory macrophages in the lung tissue and the alveoli/airways on day 7. In contrast, fewer alveolar macrophages were present in the BAL of Icam1-/-mice. The loss of body weight and the concentrations of inflammatory mediators in the BAL were also significantly greater in Icam1-/- mice. Mechanistic studies to understand the defect in clearance show that neutrophils and macrophage subpopulations had no defect in phagocytosis or acidification of phagosomes. RNA sequencing reveals many differences in gene expression, but no suggestion of a defect in phagocytosis or killing. Thus, that ICAM-1 is necessary for the clearance of S. pneumoniae and for the resolution of pneumonia, but is not required for the recruitment of neutrophils or inflammatory macrophages into the pneumonic lung parenchyma or the alveoli/airways during S. pneumoniae-induced pneumonia.

Colchicine can keep the viability of bacteria in mastitic milk by preventing leukocyte phagocytosis in dairy cow and goat.

Despite the occurrence of mastitis, no bacteria were detected in any of the milk samples after culture. This is partially because the neutrophils present in milk phagocytose bacteria during milk preservation. In this study, we investigated whether colchicine inhibited the decrease in viable bacteria in milk by suppressing phagocytosis during preservation. The number of viable bacteria decreased when cow milk was preserved for 5 h. However, the addition of 0.1 and 1% colchicine significantly increased the number of viable bacteria (p < 0.05). The percentage of culture-negative cow's milk increased more than two-fold after 5 h compared to that at 0 h of preservation, however this percentage was significantly reduced by the addition of colchicine (p < 0.05). When goat milk with mastitis was incubated with bacteria (Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus), the percentage of phagocytosed neutrophils decreased significantly with the addition of colchicine (p < 0.05). These results indicate that colchicine suppressed the decrease in the number of viable bacteria by preventing neutrophil phagocytosis during milk preservation. These findings may help in the identification of mastitis-causing bacteria and the selection of antibiotics for the treatment of mastitis.

Effects of Tooth Desensitizers on Streptococcus mutans Biofilm Formation Using a Modified Robbins Device Flow Cell System.

This study aimed to assess the antibiofilm effects of dentin desensitizers using a modified Robbins device flow cell system. The test desensitizers were Saforide, Caredyne Shield, and Clinpro White Varnish. Standardized dentin specimens were prepared from human single-rooted premolars, treated with one of the materials, and mounted on the modified Robbins device flow cell system. Streptococcus mutans biofilms were developed for 24 h at 37 °C under anaerobic conditions. Scanning electron microscopy, fluorescence confocal laser scanning microscopy, viable and total cell counts, acid production, and gene expression analyses were performed. A wavelength-dispersive X-ray spectroscopy electron probe microanalyzer was used to analyze the ion incorporations. Clinpro White Varnish showed the greatest inhibition, suggesting its suppression of bacterial adherence and transcription of genes related to biofilm formation. Saforide reduced only the number of viable bacteria, but other results showed no significant difference. The antibiofilm effects of Caredyne Shield were limited. The uptake of ions released from a material into dentin varies depending on the element. Clinpro White Varnish is effective for the short-term treatment of tooth sensitivity due to dentin demineralization. It prioritizes remineralization by supplying calcium and fluoride ions while resisting biofilm formation.

Lag Plot: A Novel Method to Determine Viable Bacterial Cell Number via a Single OD Measurement.

Bactericidal activity is a valuable parameter which is considered and measured for antimicrobial compounds. The available standard protocol to evaluate bactericidal activity is based on the direct colony count. Colony counting requires serial dilution, plating, overnight incubation, and direct counting, which is time-and labor-intensive. In regard to eliminate direct plate count, novel techniques were developed based on the real-time growth monitoring which can come with some limitations and drawbacks. These drawbacks encourage us to develop a novel technique with simple procedure to determine viable bacterial cell count. In this procedure, real-time growth monitoring is not required. In fact, after an incubation time, the number of viable bacteria can be determined with a single OD measurement and through an equation. In this regard, four standard bacterial strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Bacillus subtilis (ATCC 23857), were cultured with descending inoculum densities (1.5 × 107 to 15CFU/mL) and growth curves were drawn. As expected, growth in the samples with lower inoculum densities recorded with longer lag phase. Also, a direct relation was observed between the recorded turbidities and initial cell counts. A logarithmic curve (lag plot) was obtained by plotting the OD, after 12-18h incubation, against initial cell counts. In all examined strains, R2 was calculated in the range of 0.96-0.99 which is acceptable value for coefficient of determination. Equation corresponding to the lag plot was obtained and called lag equation. This equation is applicable to calculate the number of viable cells in unknown samples simply by inoculation, incubation, and single OD measurement. Developed technique can be introduced as a simple substitution for labor- and time-intensive direct colony counting.

The exploration of pasteurization processes and mechanisms of inactivation of Bacillus cereus ATCC 14579 using radio frequency energy

Radio frequency (RF) heating has been utilized to investigate sterilization techniques, but the mechanism of sterilization via RF heating, particularly on Bacillus cereus (B. cereus), has not been thoroughly examined. In this paper, sterilization processes and potential bactericidal mechanisms of B. cereus using RF were investigated. The best heating and sterilization efficiency was achieved at (Electrode gap 130 mm, conductivity of bacterial suspension 0.1 S/m, volume of bacterial suspension 40 mL). Heating a suspension of B. cereus to 90 °C in 80 s using RF reduced the number of viable bacteria by 4.87 logarithms. At the cellular level, there was a significant leakage of nucleic acids and proteins from the bacterial cells. Additionally, the integrity of the cell membrane was severely damaged, with a decrease in ATP concentration of 2.08 mM, Na, K-ATPase activity to 10.7 (U/109 cells), and Ca, Mg-ATPase activity to 11.6 (U/109 cells). At the molecular level, transcriptomics analysis showed that RF heating of B. cereus to 65 °C produced 650 more differentially expressed genes (DEGs) compared with RF heating to 45 °C. The GO annotation analysis indicated that the majority of differentially expressed genes (DEGs) were predominantly associated with cellular components. KEGG metabolic analysis showed enrichment in microbial metabolism in diverse environments, etc. This study investigated the potential bactericidal mechanism of B. cereus using RF, and provided some theoretical basis for the research of the sterilization of B. cereus.

Shield-armed probiotic delivery system based on co-deposition of poly-dopamine and poly-l-lysine helps Lactiplantibacillus plantarum relieve hyperuricemia

Hyperuricemia (HUA) is a disease characterized by an abnormal metabolism of purine. Lactic acid bacteria (LAB) have attracted much attention for their safe and effective treatment of HUA by inhibiting xanthine oxidase (XOD) and regulating gut microbiota. However, the effectiveness of probiotics can be compromised by the harsh environment of the gastrointestinal tract. In preliminary experiments, Lactiplantibacillus plantarum DY1, which is generally regarded as safe (GRAS), can lower uric acid. We have devised a straightforward and efficient technique for encapsulating DY1 using a coating comprising polydopamine (PDA) co-deposited with poly-l-lysine (PLL) to obtain DY1@PDLL. TEM, SEM, FT-IR and DLS tests showed that DY1 was successfully coated. Incubate at SGF or SIF for 3 h, the number of viable bacteria of free probiotics and DY1@PDLL decreased by 0.92 and 0.46 log cfu/mL, 1.66 and 0.66 log cfu/mL, respectively. The fluorescence intensity of the intestines of the DY1@PDLL treated mice was 3.96 times that of free probiotic. Notably, DY1@PDLL can reduce the uric acid levels of HUA mice by 31.63 % and free probiotics by 18.72 % (≈1.69 times). DY1@PDLL could also regulate gut microbiota and serum metabolic profile. These findings unequivocally highlight the remarkable potential of DY1@PDLL as an exceptional oral probiotic delivery system.

Comparative Analysis of Antibacterial Efficacy and Flow Characteristics of Three Root Canal Sealers.

Oral microflora containing microorganisms is responsible for the majority of orodental diseases as well as post-endodontic treatment failure. Even in the ideal root canal treatment cases, a small number of viable bacteria will remain in the dentinal tubule hence come the role of root canal sealers, which must offer and possess some degree of antimicrobial activity alongside adequate flowability to ensure excellent sealing of all lateral dentinal tubule canals to prevent the possibility of future reinfection. The present study aimed to examine the antibacterial efficacy against Enterococcus faecalis (E. faecalis) using an agar diffusion test (ADT) at different time intervals, as well as the flow characteristics of three different root canal sealers: Endofill (Produits Dentaires SA,Vevey, Switzerland), AH Plus (Dentsply De Trey GmbH, Konstanz, Germany), and the newly introduced GuttaFlow2 (Coltène/Whaledent, Altstätten, Switzerland). The antibacterial activity and flow characteristics of three root canal sealers, Endofill, AH Plus, and GuttaFlow2, were tested using ADT at three different time intervals (24 hours, 48 hours, and seven days). For this purpose,E. faecalis strains were used as the pathogen in this study. Flow characteristics were done using the standard flow test protocol recommended for endodontic sealers. Data were analyzed using one-way ANOVA and the Tukey post hoc test, with a p-value < 0.05 considered statistically significant. The results for antibacterial activity showed a statistically significant difference between Endofill and the other groups (p < 0.05). The antibacterial activity of Endofill increased over time from 24 hours to 48 hours and seven days. AH Plus demonstrated antibacterial activity only within the first 24 hours of mixing, while GuttaFlow2 showed no inhibition zones against E. faecalis. Regarding the flow test results, the Endofill group recorded the lowest flow values compared to GuttaFlow2 and AH Plus, which was statistically significant (p < 0.05). There was no statistically significant difference between GuttaFlow2 and AH Plus for flow values. Endofill demonstrated the highest antibacterial activity at all time intervals, while GuttaFlow2 showed no antimicrobial activity. AH Plus exhibited antimicrobial effects only within the first 24 hours of mixing. In terms of flow values, Endofill had the lowest flow, whereas GuttaFlow2 and AH Plus had the highest flow values.

Kinetic study of the thermal inactivation of Weizmannia coagulans during food thermal processing

Weizmannia coagulans has attracted attention due to its remarkable health benefits for human, but the dynamic changes of its viable bacteria during thermal processing have been less reported. In this study, a predictive model for the survival of Weissmanella coagulans during thermal processing of food was developed and validated during the processing of coffee, tea, instant noodles, calcium milk biscuits, muffin cake and steamed buns. The kinetics of heat inactivation activities of Weizmannia coagulans VHProbi C08 and Weizmannia coagulans GBI-30, 6086 at 85, 95, 105, 110 and 115 °C were investigated, and their coefficients of determination were greater than 0.91 and 0.87, and the root-mean-square errors were less than 0.64 and 0.43, respectively. The z-values of VHProbi C08 and GBI-30, 6086 were obtained by Bigelow model fitting as 36.1 °C and 36.9 °C, respectively. The developed prediction model was applied to the thermal processing of six food products and the measured values were all within ±0.5 Log10 (CFU/mL) of the predicted values, indicating high prediction accuracy. The model predicts the survival of Weissmanella coagulans simply by obtaining the initial number of viable bacteria and the change in temperature. These suggested that the model can be used as an effective tool to evaluate the stability of Weizmannia coagulans in food thermal processing.

Synergistic effects of arginine and fluoride on human dental biofilm control

ObjectivesThe aim of this study was to quantitatively and comprehensively investigate the combined effects of arginine and fluoride on the suppression of pathogenicity using an in situ biofilm model and next-generation sequencing (NGS). MethodsUsing the in situ model, dental biofilms were formed and the viable bacterial counts and arginine activity in the arginine- and fluoride-containing dentifrice and control groups were measured. We also compared their effects on the bacterial microbiota and predictive functional factors in the control, arginine (arg), and arginine + fluoride (argF) groups using NGS analysis. ResultsCompared to the control treatment, the use of 8 % arginine and 1450 ppm fluoride toothpaste resulted in significantly high oral NH4+ concentrations without affecting the number of viable bacteria (P < 0.05). NGS analysis revealed that the oral microbiota of the control, arg, and argF groups were significantly different. Heat map analysis of the predicted functional factors revealed that the arg group had different properties from the other groups and activated specific substrate metabolic pathways; contrastingly, argF treatment inhibited the activity of these pathways and prevented an increase in the abundance of bacterial genera that utilize substrates such as sucrose, suggesting the synergistic effect of arginine and fluoride. ConclusionsThis study indicates that the combination of arginine and fluoride has a synergistic effect on the bacterial microbiota and pathogenicity of dental biofilms compared with arginine alone. Clinical significanceOur findings suggest that the combination of arginine and fluoride could be used as an effective prebiotic and may inhibit the growth of bacteria associated with dental diseases.

Farnesol Emulsion as an Effective Broad-Spectrum Agent against ESKAPE Biofilms.

The family of ESKAPE pathogens is comprised of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter. Together they are the main contributors of nosocomial infections and are well established for their ability to "escape" antibiotics. Farnesol is an FDA-approved cosmetic and flavoring agent with significant anti-biofilm properties. In a proprietary emulsion, farnesol has been shown to be capable of disrupting S. aureus, P. aeruginosa, and A. baumannii biofilms. The current work demonstrates that this farnesol emulsion reduces the number of viable bacteria, while also leading to reductions in biomass, of the other three ESKAPE pathogens: Enterococcus faecium, Klebsiella pneumoniae, and Enterobacter, both in vitro and in an ex vivo human skin model. A concentration of 0.5 mg/mL was effective for impeding biofilm development of all three bacteria, while 1 mg/mL for E. faecium and K. pneumoniae, or 0.2 mg/mL for E. cloacae, was able to kill bacteria in established biofilms. Contrary to antibiotics, no resistance to farnesol was observed for E. faecium or K. pneumoniae. The results indicate that farnesol is effective for direct cell killing and also has the ability to induce biofilm detachment from surfaces, as confirmed using Live/Dead image analysis. Our findings confirm that farnesol emulsion is an effective broad-spectrum agent to impede ESKAPE biofilms.

Efficiency of the photodynamic therapy on viability of Streptococcus mutans in the oral cavity using chitosan nanoparticles: an in vitro study.

This study aimed to investigate the efficiency of antimicrobial photodynamic therapy (aPDT) on Streptococcus mutans biofilm in the oral cavity using the photosensitizer chloroaluminum phthalocyanine encapsulated in chitosan nanoparticles (ClAlPc/Ch) at three preirradiation times. Biofilms of Streptococcus mutans strains (ATCC 25,175) were cultivated on bovine tooth blocks and exposed to a 10% sucrose solution three times a day for 1min over three consecutive days. The samples were randomly distributed into five treatment groups (n = 5): (I) aPDT with ClAlPc/Ch with a preirradiation time of 5min (F5), (II) aPDT with ClAlPc/Ch with a preirradiation time of 15min (F15), (III) aPDT with ClAlPc/Ch with a preirradiation time of 30min (F30), (IV) 0.12% chlorhexidine digluconate (CHX), and (V) 0.9% saline solution (NaCl). After treatment, the S. mutans biofilms formed on each specimen were collected to determine the number of viable bacteria (colony-forming units (CFU)/mL). Data were analyzed for normality using the Shapiro-Wilk test and the analysis of variance (ANOVA) and Tukey HSD tests to analyze the number of viable bacteria (α = 0.05). The one-way ANOVA showed a difference between the groups (p = 0.0003), and the Tukey HSD posttest showed that CHX had the highest microbial reduction of S. mutans, not statistically different from the F5 and F15 groups, whereas the NaCl group had the lowest microbial reduction statistically similar to the F30 group. The results demonstrate that aPDT mediated by ClAlPc/Ch when used at preirradiation times of 5-15min can be an effective approach in controlling cariogenic biofilm of S. mutans, being an alternative to 0.12% CHX.

Production and preservation quality assessment of canned beef

The present study was undertaken to produce canned beef along with the determination of its preservation quality. Kalojira oil (Nigella sativa), Na-nitrite (NaNO2 ) and common salt (NaCl) were added in beef and treated as preservative groups namely T1 , T2 and T3 treatment group respectively. Beef without addition of any preservative kept as control group (T0 ) and each group or treatment containing three (03) replications. Data from both fresh raw beef and canned beef were compared statistically in an ANOVA of a Completely Randomized Design using General Linier Model Procedures of SPSS, 20 computer software packages. Beef was purchased from local market immediate after slaughter and brought at meat processing laboratory of BLRI. The physicochemical properties and microbiological properties of both raw beef and canned beef were recorded. The physical properties viz, pH, drip and cook loss of fresh raw beef were 6.40, 5.55% and 27.71%, respectively. The moisture and CP content in raw beef were 74.61 and 25.39%, respectively. The total viable bacteria count and Coliform count in raw beef were 8.8 × 106 and 4.3 × 104 cfu/g, respectively. The pH of canned beef was higher (p &lt; 0.05) in meat preserved with NaNO2 and Kalojira oil than that of NaCl and control group. Preservatives, however, had no effects (p &gt; 0.05) on moisture or DM and CP content in canned beef. Though, there was no significant (p &gt; 0.05) effect, but Kalojira oil performed as impressive preservative compared to others. In microbiological aspects of canned meat, the TVC, TCC, Salmonella spp. and Staphylococcus spp. were entirely absent in Kalojira group. In case of NaNO2 and NaCl group, only a very few numbers of viable bacteria were found (1 × 102 ). On the other hand, only the Staphylococcus bacteria were found in case of control group. Considering the physico-chemical and microbiological aspects in canned beef, it may be concluded that the Kalojira oil could be a suitable value-added preservative in meat canning.

Preparation of bacterial fertilizer from biogas residue after anaerobic co-digestion of kitchen waste and residual sludge.

Azotobacter chroococcum and Bacillus subtilis were selected as fermentation strains, and biogas residue after anaerobic digestion of kitchen waste and residual sludge was used as fermentation substrate. A single factor optimization test was used to optimize the solid-state fermentation parameters of biogas residue with the number of viable bacteria and the number of spores as indexes. The results showed that the optimum inoculation conditions involved the following: 55% initial moisture content, 15% initial inoculation amount, 30 ℃, and 1:1 initial inoculation ratio for 13days. Pot experiment showed that the prepared three kinds of bacterial fertilizers could not only effectively promote the growth of white clover, improve the composition of soil nutrients, but also change the structure of soil bacterial community, which is of great significance to the health of soil ecosystem in white clover.

In vitro and in vivo antimicrobial activity of the fumigant, ethyl formate, against bacteria and fungi of date fruits in postharvest phase

The search is on for safer alternatives to fumigants like methyl bromide and phosphine, which harm the ozone layer and pose other risks. Ethyl formate is a promising candidate due to its potential antimicrobial and insecticidal action in stored products like date fruits. The susceptibility of date fruits to microbial spoilage is significantly influenced by factors such as moisture content, storage practices, and environmental conditions. In the present study, the in vitro antimicrobial effect of ethyl formate against three microbial species, namely Aspergillus niger F4, Mucor circinelloides YMM22, and Pseudomonas aeruginosa B1 was tested under laboratory conditions. In addition, two common date fruit varieties, semi-dry El-Wady I and the dry Frehi, were fumigated with ethyl formate alone in a laboratory-scale prototype and with a mixture of ethyl formate and CO2 at a semi-industrial scale prototype. Both varieties had an initial moisture content of around 13.40 and 12.48% for El-Wady I and Frehi, respectively. The fumigation was conducted using the optimal concentration of 70 mg/L air for 24 h incubation period. Fumigation with ethyl formate alone or in combination with CO2 significantly reduced the number of viable bacteria and fungi (CFU/g), but the potential for a combined effect with CO2 was not investigated. The percentages of reduction in the fungal count were 78.18 and 90.76% for Frehi and El-Wady I varieties, respectively in a semi-industrial scale prototype. Moreover, the percentages of reduction in the bacterial count were 53.31 and 65.20%, respectively for Frehi and El-Wady I varieties. Ethyl formate showed promise as a natural alternative to control microbial contamination of stored date fruits. Further research suggests that increasing fumigation duration or concentration could provide comprehensive control of microbial pathogens at all stages of development.

Effect of baicalin on eradicating biofilms of bovine milk derived Acinetobacter lwoffii

BackgroundAcinetobacter lwoffii (A.lwoffii) is a serious zoonotic pathogen that has been identified as a cause of infections such as meningitis, bacteremia and pneumonia. In recent years, the infection rate and detection rate of A.lwoffii is increasing, especially in the breeding industry. Due to the presence of biofilms, it is difficult to eradicate and has become a potential super drug-resistant bacteria. Therefore, eradication of preformed biofilm is an alternative therapeutic action to control A.lwoffii infection. The present study aimed to clarify that baicalin could eradicate A.lwoffii biofilm in dairy cows, and to explore the mechanism of baicalin eradicating A.lwoffii.ResultsThe results showed that compared to the control group, the 4 MIC of baicalin significantly eradicated the preformed biofilm, and the effect was stable at this concentration, the number of viable bacteria in the biofilm was decreased by 0.67 Log10CFU/mL. The total fluorescence intensity of biofilm bacteria decreased significantly, with a reduction rate of 67.0%. There were 833 differentially expressed genes (367 up-regulated and 466 down-regulated), whose functions mainly focused on oxidative phosphorylation, biofilm regulation system and trehalose synthesis. Molecular docking analysis predicted 11 groups of target proteins that were well combined with baicalin, and the content of trehalose decreased significantly after the biofilm of A.lwoffii was treated with baicalin.ConclusionsThe present study evaluated the antibiofilm potential of baicalin against A.lwoffii. Baicalin revealed strong antibiofilm potential against A.lwoffii. Baicalin induced biofilm eradication may be related to oxidative phosphorylation and TCSs. Moreover, the decrease of trehalose content may be related to biofilm eradication.

The microbiome of the pregnant uterus in Holstein dairy heifers and cows assessed by bacterial culture and 16S ribosomal RNA gene sequencing.

The possibility that there is a resident and stable commensal microbiome within the pregnant uterus has been supported and refuted by a series of recent studies. One element of most of the initial studies was that they were based primarily on 16S rRNA gene sequencing from bacteria. To account for this limitation, the current study performed both bacterial culture and 16S rRNA gene sequencing in a side-by-side manner (e.g., same tissues isolated from the same animal). The uteruses of 10 mid-pregnant (156 ± 5 d of gestation) Holstein heifers and cows were collected following slaughter. The external surface of the reproductive tract (positive control for contamination during tissue collection) as well as tissues within the pregnant uterus (placentome, inter-cotyledonary placenta, inter-caruncular endometrium, amnionic fluid, allantoic fluid, fetal abomasum content, and fetal meconium) were sampled for bacterial culture and 16S rRNA gene sequencing. There were 87 unique bacterial species cultured from the external surface of the pregnant reproductive tract (contamination control) and 12 bacterial species cultured from pregnancy tissues. Six out of 10 cattle (60%) exhibited bacterial growth in at least one location within the pregnant uterus. For the metataxonomic results (16S rRNA gene sequencing), a low targeted microbial biomass was identified. Analyses of the detected amplicon sequence variants (ASV) revealed that there were: (1) genera that were prevalent on both the external surface and within the pregnant uterus; (2) genera that were prevalent on the external surface but either not detected or had very low prevalence within the pregnant uterus; and (3) genera that were either not detected or had low prevalence on the external surface but found with relatively high prevalence within the pregnant uterus. There are a small number of viable bacteria in the pregnant uterus. The 16S rRNA gene sequencing detected a microbial community within the pregnant uterus but with a low biomass. These results are consistent with recent studies of the pregnant bovine uterus and leave open the question of whether there is adequate microbial mass to significantly affect the biology of the normal healthy bovine pregnancy.

Flow cytometry: Unravelling the real antimicrobial and antibiofilm efficacy of natural bioactive compounds

Flow cytometry (FCM) provides unique information on bacterial viability and physiology, allowing a real-time early warning antimicrobial and antibiofilm monitoring system for preventing the spread risk of foodborne disease. The present work used a combined culture-based and FCM approach to assess the in vitro efficacy of essential oils (EOs) from condiment plants commonly used in Mediterranean Europe (i.e., thyme EO, oregano EO, basil EO, and lemon EO) against planktonic and sessile cells of food-pathogenic Listeria monocytogenes 56 LY, and contaminant and alterative species Escherichia coli ATCC 25922 and Pseudomonas fluorescens ATCC 13525. Evaluation of the bacterial response to the increasing concentrations of natural compounds posed FCM as a crucial technique for the quantification of the live/dead, and viable but non-culturable (VBNC) cells when antimicrobial agents exert no real bactericidal action. Furthermore, the FCM results displayed higher numbers of viable bacteria expressed as Active Fluorescent Units (AFUs) with a greater level of repeatability compared with outcomes of the plate-count method. Overall, accurate counting of viable microbial cells is a critically important parameter in food microbiology, and flow cytometry provides an innovative approach with high-throughput potential for applications in the food industry as “flow microbiology”.

Effect of the Addition of Dandelion (Taraxacum officinale) on the Protein Profile, Antiradical Activity, and Microbiological Status of Raw-Ripening Pork Sausage.

The study evaluated the effect of adding dandelion extract on the characteristics of raw-ripening pork sausages while reducing the nitrite addition from 150 to 80 mg/kg. The sausages were made primarily from pork ham (80%) and pork jowl (20%). The process involved curing, preparing the meat stuffing, forming the links, and then subjecting the sausages to a 21-day ripening period. Physicochemical parameters such as pH, water activity, and oxidation-reduction potential were compared at the beginning of production and after the ripening process. The study also examined the impact of ripening on protein metabolism in pork sausages and compared the protein profiles of different sausage variants. The obtained research results indicate that dandelion-leaf extract (Taraxacum officinale) were rich in phenolic acids, flavonoids, coumarins, and their derivatives (LC-QTOF-MS method). Antiradical activity test against the ABTS+* and DPPH radical, and the TBARS index, demonstrated that addition of dandelion (0.5-1%) significantly improved the oxidative stability of raw-ripening sausages with nitrite content reduction to 80 mg/kg. A microbiological evaluation of the sausages was also carried out to assess the correctness of the ripening process. The total number of viable bacteria, lactic acid bacteria, and coliforms were evaluated and subsequently identified by mass spectrometry.

Antibacterial activity of closantel against methicillin-resistant Staphylococcus aureus and itsbiofilm.

The antimicrobial resistance of Staphylococcus aureus (S. aureus) has become a challenge in the treatment of infectious diseases. It is of great clinical value to discovery effective antimicrobial agents against multi-drug resistant S. aureus and its biofilms. This study aims to explore the antibacterial activity of the antiparasitic drug closantel against methicillin-resistant S. aureus and its biofilms through drug repurposing. The sensitivity of S. aureus to closantel was assessed using microbroth dilution and disk diffusion methods. The bacteriostatic and bactericidal activities of closantel were determined by time-kill curves and colony count. Scanning electron microscopy combined with SYTOX Green and DiSC3(5) fluorescence probes were used to study the bactericidal mechanism of closantel. The influence of resistance was assessed by continuous exposure to sub-inhibitory concentrations of closantel. The anti-biofilm activity was evaluated using 96-well plates and crystal violet staining, and cytotoxicity was measured using the CCK-8 assay. The minimal inhibitory concentration (MIC) of closantel for both methicillin-sensitive and methicillin-resistant S. aureus ranged from 0.125 to 1.000 μg/mL. Disk diffusion tests showed that 80 μg of closantel created an inhibition zone, which increased in diameter with higher drug amounts. Sub-inhibitory concentrations (0.031 μg/mL) of closantel significantly inhibited S. aureus proliferation, reducing bacterial turbidity from 0.26±0.00 to 0.11±0.01 (t=16.06, P<0.001), with stronger inhibition at higher concentrations. Closantel at 0.25×MIC inhibited S. aureus proliferation for 12 hours, while 1×MIC inhibited it for over 24 hours, with the number of viable bacteria decreasing as the drug concentration increased. Mechanistic studies indicated that closantel effectively disrupted the integrity of S. aureus cell membranes, significantly increasing SYTOX Green and DiSC3(5) fluorescence intensity. Even after 25 days of continuous exposure to sub-inhibitory concentrations of closantel, no resistance developed. Closantel at 0.0625 μg/mL significantly inhibited biofilm formation, reducing it from 1.29±0.16 to 0.62±0.04 (t=11.62, P<0.001), showing a clear dose-dependent effect. Closantel at 2 μg/mL also significantly eradicated established biofilms, reducing biofilm mass from 1.62±0.34 to 0.51±0.39 (t=4.84, P<0.01). Additionally, closantel exhibited extremely low cytotoxicity, with half-maximal lethal concentrations for HepG2 liver cancer cells and normal LO2 liver cells both exceeding 64 μg/mL. Closantel exhibits strong antibacterial activity against S. aureus and its biofilm with low cytotoxicity against human cells, making it a promising candidate for new therapeutic strategies against S. aureus-related infections.